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VesiclesDOI: 10.1002/ange.201004003
Triggered Templated Assembly of Protein Polymersomes**Feng Li, Frits A. de Wolf, Antonius T. M. Marcelis, Ernst J. R. Sudh�lter,Martien A. Cohen Stuart, and Frans A. M. Leermakers*
There is a strong demand for robust targetable nanosizedcontainers in biomedical applications. The development of ageneral strategy to assemble functional biomolecules intostable nanostructures with desired size and shape is challeng-ing. Liposomes for instance are extremely fragile and havelow stimuli responsiveness and chemical diversity.[1] Mostpolymersomes, on the other hand, lack biofunctionality, whichrestricts their ability to interact with cells or tissues. Herein wepresent a versatile method to make stable biocompatibleprotein polymersomes by a triggered templated self-assemblyroute. Pluronic vesicles (see Figure 1), routinely fabricatedwith a narrow size distribution that ranges from 50–2000 nmin diameter, serve as a matrix that can take up large quantitiesof biosynthetic triblock copolymers CSXSXC in their unila-mellar shell. The middle block SXSX of the protein has silk-like repeats, where X stands for the chargeable amino acidsglutamic acid (E) or histidine (H). In response to a change ofthe pH value (down or up, respectively), the S block of theprotein polymers becomes hydrophobic and inserts into thetemplate, thus leading to the formation of protein polymer-somes. Alternatively, by adding negatively charged CSESEC orsiRNA to the positively charged CSHSHC, co-assembly andco-insertion occurs at neutral pH values. The C block formsthe stabilizing corona, is collagen-like, and has been shown tobe hypoallergenic. Hence, biocompatible[2] multifunctionalcapsules for drug- and gene-delivery applications areobtained.
Our method may be characterized as “triggered tem-plated assembly” (TTA). In a first step, we fabricate medicallycompatible Pluronic vesicles[3] with tunable diameters in therange of 0.1–2 mm in the presence of fully water-solubleprotein CSXSXC polymers (Figure 1).[4] In a second step, wetrigger the assembly by neutralizing the charge X on theS block. The protein adopts a b-sheet secondary structuresand inserts itself into the vesicle walls. We refer to thesecapsules as protein polymersomes. Polymersomes based onpolypeptides have received a great deal of interest in recentyears.[5–7] Typical examples of protein polymersomes are
vesicles made of chemical synthetic polypeptide blockcopolymers[2, 5] or virus capsides.[7] These protein polymer-somes generally have excellent biocompatibility, but onedrawback is that it is rather tedious and time-consuming tomake the functionalized starting material. Our TTA system,however, overcomes this problem.
All the molecules for the TTA system are available inlarge amounts. The primary vesicles that form the templatematerial are made of Pluronic L121 (PEO5–PPO68–PEO5),which is a thermosensitive amphiphilic block copolymer witha large, marginally hydrophobic poly(propylene oxide)middle block and two very short oligo(ethylene oxide) outerblocks.[4] This polymer spontaneously forms rather unstableunilamellar vesicles in a small temperature window: below15 8C the polymer is molecularly dissolved and above 25 8Cthe bare vesicles quickly aggregate. Around room temper-ature the fragile membrane is composed of loosely packedpolymers and acts as the template ready to host thepolypeptide block copolymers. These peptides give stabilityto the otherwise fragile polymersomes.
Herein we employ designed protein block copolymers, inwhich chosen amino acid sequences are expressed in anappropriate host organism. With this method, absolutecontrol over the polymer length and sequence provided by abiosynthetic approach is retained, and biocompatible prod-ucts with large quantities and unprecedented specificationsare produced.[8] Two such protein polymers, which may be
Figure 1. Triggered templated assembly of protein polymersomes.Pluronic L121 vesicles (light red core with thin blue corona) sur-rounded by triblock peptide copolymer CSXSXC (S block: red, C block:blue). After a trigger, the X groups (spherical dots) become uncharged.The S blocks adopt a b-sheet secondary structure, the hydrophobicityof which drives the insertion of the protein polymers into the capsule.
[*] F. Li, Dr. F. A. de Wolf, Dr. A. T. M. Marcelis,Prof. Dr. M. A. Cohen Stuart, Prof. Dr. F. A. M. LeermakersDepartment of Agriculture and Food ScienceDreijenplein 8, 6703 HB Wageningen (The Netherlands)
Prof. Dr. E. J. R. Sudh�lterDepartment of Chemical EngineeringJulianalaan 136, 2628 BL Delft (The Netherlands)
[**] This research is supported by NanoNed, a national nanotechnologyprogram coordinated by the Dutch Ministry of Economic Affairs.
Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201004003.
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recognized as twins because they have an almost identicalprimary sequence of amino acids, are applied in our study.Both molecules were separately expressed in yeast, and thetwins have a modular CSXSXC structure. The C block carriesfew charges and is water-soluble at all pH values used. Thecentral motive SX has a number of silk-like repeats separatedby a chargeable amino acid X, which differ for the twincompounds (histidine: CSHSHC, glutamic acid: CSESEC). Ifthe groups are charged (neutral pH) the S block has nosecondary structure and is water-soluble (Figure 1). However,in the absence of the charge, folding takes place and b rolls orin some cases b sheets form through intramolecular hydro-gen-bonding.[9] These secondary structure elements havehydrophobic faces and they may further assemble intoribbonlike aggregates in a rather slow “nucleation andgrowth” process that can easily take several hours (dependingon the actual protein concentration). Extensive investigationsof these objects were recently reported.[8,10] A key observationis that one dimension of the ribbon, namely the distance thatseparates the two collagen-like blocks, is approximately12 nm. This dimension is compatible with the PPO block ofthe Pluronic vesicle (Figure 1). Therefore it is reasonable toassume that hydrophobic interactions drive these proteinpolymers to enter the Pluronic vesicle membrane once thecharges in the S block are neutralized.
Indeed, under such conditions, the protein polymersreadily associate with the vesicle template and then formprotein polymersomes. For facile imaging of the protein, wefluorescently labeled the C blocks of the protein polymers.Fluorescein isothiocyanate (FITC) dyes were exclusivelyattached to the lysine amino group by isothiocyanatecoupling, and the labels do not interfere with the drivingforce for the self-assembly of the protein polymers. Resultsfor giant polymersomes stabilized by labeled protein poly-mers CSESEC are shown in Figure 2. Upon pH triggering,remarkably monodisperse vesicles are clearly visible from theconfocal microscopy image. In this particular example, wealso added the hydrophobic fluorescent label Nile red, whichhighlights the hydrophobic regions (Figure 2a). The locationof FITC-labeled proteins is shown in Figure 2b. The overlayof the images (Figure 2c) proves that, at the resolution of theoptical microscope, most of the protein polymers are at thesame location as the Pluronic vesicles.
We realized that the fragile Pluronic vesicles can beextruded through a polycarbonate membrane and then adopta smaller size. The triggered assembly of protein polymers in
extruded polymersomes gives relatively monodisperse pro-tein polymersomes with a controlled diameter of, for exam-ple, up to 100 nm (see Figure S1 in the Supporting Informa-tion). Dynamic light scattering (DLS) shows that the size iskept constant for at least two weeks (Figure 3). A very small
trend in the data indicates that the average size of the vesiclesincreases and also the intensity of the scattered light growsslowly. This observation is consistent with a slow addition ofprotein polymers to the vesicle membrane over time.Interestingly, the Contin software analysis shows that thevesicles become progressively more monodisperse.
It is important to note that the protein polymers are mostlikely not simply molecularly dissolved in the hydrophobicvesicle membrane phase. All protein polymers are extremelyidentical, chirally pure, and monodisperse. These uniqueproperties give the protein polymers the extraordinary abilityto assemble into intermolecular aggregates. Such proteinordering is expected to continue until very large protein toPluronic ratios are reached. Small angle neutron scattering(SANS) measurements confirm that the thickness of thebilayer core significantly increases from 6 nm to 16 nm withthe addition of 2 mgmL�1 CSESEC; the thickness of thebilayer core even reaches 20 nm after addition of another0.25 mg mL�1 CSHSHC (Figure 4). The change in bilayer corethickness indicates that the silk-like domain is inserted intothe Pluronic vesicle membrane, while keeping the collagen-like domains in the aqueous phases. Indeed, the build-up ofthese layers is assumed to be responsible for the improvedstability of the capsules.
The time needed for the protein polymers to be insertedinto the vesicle membrane is apparently shorter than thatneeded for protein self-assembly into long rigid ribbons. Theinstant stabilization of Pluronic vesicles also indicates that theinsertion process is fast. As proven by CD spectroscopy (seeFigure S3 in the Supporting Information), the protein poly-mers adapt themselves to the polymersome structure byintramolecular hydrogen-bonding, b sheet, or b rolls secon-dary structures. By using fluorescence correlation spectros-copy (FCS), we could quantitatively determine the speed and
Figure 2. Confocal microscopic images of: a) vesicle membrane la-beled with Nile red; b) CSESEC labeled with FITC; c) overlapping of thefirst two images. Scale bar: 5 mm.
Figure 3. DLS stability study of 100 nm extruded vesicles. Top curve(relating to right ordinate): hydrodynamic radius of vesicles bycumulant fitting; bottom curve (relating to left ordinate): scatteredintensity of the vesicles; the inset shows Contin software analysisresults, 24 h after preparation (gray curve) and 280 h after preparation(black curve).
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the amount of protein polymers that participate in the build-up of the vesicle membrane (Figure 5). Pluronic vesicles areinitially permeable for the protein polymer; no protein is
detected on the vesicle membrane directly after samplepreparation. However, roughly 40 % of the overall amount ofprotein participates in the formation of the vesicle membranewithin four hours, and the incorporation of proteins levels offat approximately 70% seven hours after sample preparation.
This new method to fabricate stable capsules is versatile inthe sense that we can incorporate various biologically activematerials into the protein polymersome by selection of thetriggering method. For instance, the positively chargedprotein polymer CSHSHC (Figure 6a–c) can be triggered byaddition of negatively charged polyelectrolytes, such assiRNA (Figure 6g–h), which then makes a stable biocompat-ible gene container. In this case, the biologically active speciesis also responsible for the stability of the capsules (and givesthe capsules a cooperative release mechanism). This approachpresents many possibilities to assemble multifunctionalbiomolecules into stable nanostructures with desired sizeand shape. Moreover, the protein polymersome that containsboth CSESEC and CSHSHC polymer is delivered into humancells, because both protein species are taken up by thecytoplasm of the cell (Figure 6d–f). No significant cell deathwas observed under the utilized experimental conditions. Thisresult suggests that biomolecules incorporated into thesevesicles can easily be transported in vivo. Note that effective
in vivo application of siRNA leads to various noveltherapeutic approaches.[11]
Since the protein polymer CSXSXC is expressed in yeastwe easily can produce large quantities (grams) of proteins.This possibility is a significant improvement over syntheticpolypeptides. In addition, other functional groups can bedesigned and incorporated into the protein polymer, if they
are either produced in yeast or as a post-chemical modifica-tion. For example, an appropriate targeting sequence can beinserted in the C block of the protein polymers, thus allowingthe capsules to deliver their contents in a very controlled way.Our TTA method leads to new multifunctional capsules fordrug delivery and gene-delivery applications.
Received: July 1, 2010Published online: November 23, 2010
.Keywords: block copolymers · drug delivery · gene delivery ·protein polymersome · vesicles
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Figure 4. SANS measurements of 100 nm extruded vesicles with differ-ent proteins.[12] a) From top to bottom: 8 mgmL�1 L121 with2 mgmL�1 CSESEC (dark gray), 2 mgmL�1 CSESEC and 0.25 mgmL�1
CSHSHC (light gray). b) Expansion of the marked area in (a).
Figure 5. FCS measurements of 100 nm extruded vesicles with FITC-labeled CSESEC. a) Autocorrelation curve measured at different times aftersample preparation. b) Amount of protein on polymersome bilayer as afunction of time. Numbers were subtracted from two-component modelfitting. The solid exponential curve was plotted to guide the eye.
Figure 6. Coassembly of protein polymersome and their delivery intoliving cells. Capsules with both negatively and positively chargedprotein polymers: a) CSHSHC labeled with FluorTM647; scale bar: 5 mm.b) CSESEC labeled with FITC. c) Overlay of the first two images. Uptakeof capsules with r = 100 nm stabilized by CSHSHC and CSESEC in caco-2cells. The protein polymersomes enter the cytoplasm not the nucleus.d) Color of FluorTM647 labeled CSHSHC, scale bar: 10 mm. e) Color ofFITC labeled CSESEC. f) Overlay of the images (d) and (e). Coassemblyof Pluronic vesicles with CSHSHC and siRNA. g) CSHSHC labeled withFluorTM647; scale bar: 2 mm. h) FITC-labeled siRNA. i) Overlay of theimages (g) and (h). See Figure S4 in the Supporting Information forimages with more cells.
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[12] http://kur.web.psi.ch/sans1/SANSSoft/sasfit.html.
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10146 www.angewandte.de � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2010, 122, 10143 –10146